郭敏 余鵬飛 白檳 邱兆巖 王謙 趙青川 李樹鈞
·論著·
細(xì)胞凋亡通路在小鼠酒精性胰腺炎發(fā)展過程中的作用
郭敏 余鵬飛 白檳 邱兆巖 王謙 趙青川 李樹鈞
目的 探討細(xì)胞凋亡通路在酒精性胰腺炎中的作用。方法 將C57BL/J小鼠隨機(jī)分為對(duì)照組(NC)、酒精組(AC)、急性胰腺炎組(AP)和酒精性胰腺炎組(AAP)。飲酒精方案為10%乙醇喂養(yǎng)2 d,15%乙醇喂養(yǎng)5 d,20%乙醇喂養(yǎng)直到13周。采用腹腔注射雨蛙素50 μg/kg體重,連續(xù)7次,每次間隔1 h的方法制備AP模型。取血檢測(cè)血清淀粉酶、脂肪酶活性;取部分胰腺稱濕、干重,計(jì)算胰腺組織水含量;取部分胰腺組織常規(guī)行病理學(xué)檢查;采用蛋白質(zhì)印跡法檢測(cè)胰腺組織凋亡相關(guān)蛋白caspase3、caspase8表達(dá),TUNEL法檢測(cè)胰腺組織的細(xì)胞凋亡。結(jié)果 NC組、AC組、AP組、AAP組小鼠血清淀粉酶活性分別為(3 630±259)、(3 196±187)、(35 955±4 607)、(53 607±3 848)U/L,脂肪酶活性為(502±41)、(745±42)、(7 346±665)、(12 764±2 544)U/L,胰腺水含量為(70.2±3.1)%、(69.6±2.0)%、(78.2±1.5)%、(85.0±3.0)%,胰腺組織caspase 3表達(dá)量為1.017±0.078、1.287±0.097、2.018±0.078、0.244±0.024,caspase 8表達(dá)量為0.829±0.010、0.599±0.074、1.270±0.080、0.145±0.015,凋亡細(xì)胞數(shù)為1、6、214、97個(gè)/10倍視野,胰腺病理評(píng)分為0、0、(7.0±0.4)、(12.8±0.3)分。AP組的血清淀粉酶、脂肪酶、胰腺水含量、胰腺病理評(píng)分均顯著高于NC組,AAP組又均顯著高于AP組,差異有統(tǒng)計(jì)學(xué)意義(P<0.05或<0.01);AP組的caspase3、caspase 8表達(dá)量、細(xì)胞凋亡數(shù)均顯著高于NC組,而AAP組的這些指標(biāo)均顯著低于AP組,且caspase3、caspase8表達(dá)量均顯著低于NC組,差異有統(tǒng)計(jì)學(xué)意義(P值均<0.05)。結(jié)論 長(zhǎng)期飲用酒精可加重胰腺炎的嚴(yán)重程度,其機(jī)制可能是通過抑制細(xì)胞凋亡,促進(jìn)腺泡細(xì)胞壞死所致。
胰腺炎,酒精性; 乙醇; 細(xì)胞凋亡; 小鼠
急性胰腺炎(AP)是一種由腺泡細(xì)胞損傷起始的胰腺外分泌的炎癥反應(yīng),其病理特征為腺泡細(xì)胞內(nèi)酶原的活化、炎癥細(xì)胞的浸潤(rùn)、腺泡細(xì)胞內(nèi)空泡化和胰腺組織的壞死[1]。一般來說,80%的AP表現(xiàn)為輕癥(MAP)或中度重癥(MSAP),其余患者會(huì)發(fā)展為重度急性胰腺炎(SAP),伴隨著全身多臟器的損傷,包括SIRS 、休克、ARDS 、腎臟衰竭等[2]。酒精是除膽結(jié)石之外AP最常見的病因之一[3]。有研究報(bào)道,長(zhǎng)期飲酒可加重胰腺炎的嚴(yán)重程度,酗酒者更易出現(xiàn)其他器官并發(fā)癥[4]。本研究旨在探討細(xì)胞凋亡通路在酒精性胰腺炎發(fā)展過程的作用。
一、實(shí)驗(yàn)動(dòng)物及分組
20只8周齡C57BL/J小鼠,體重18~20 g,購(gòu)于第四軍醫(yī)大學(xué)實(shí)驗(yàn)動(dòng)物中心。適應(yīng)環(huán)境1周后,按數(shù)字表法隨機(jī)分為對(duì)照組(NC)、酒精組(AC)、急性胰腺炎組(AP)和酒精性胰腺炎組(AAP),每組5只。NC組給予正常飲水;AC組給予10%乙醇喂養(yǎng)2 d,15%乙醇喂養(yǎng)5 d,20%乙醇喂養(yǎng)直到13周[5];AP組采用腹腔注射雨蛙素(美國(guó)Sigma公司)50 μg/kg體重,連續(xù)7次,間隔時(shí)間1 h的方法制備AP模型;AAP組則在AC組的飲酒方案基礎(chǔ)上制作AP模型。在最后一次雨蛙素注射后1 h處死小鼠。通過眼球取血標(biāo)本,同時(shí)收集胰腺組織,部分甲醛固定,部分液氮保存。
二、方法
1.血清淀粉酶、脂肪酶活性檢測(cè):應(yīng)用全自動(dòng)生化分析儀檢測(cè)小鼠血清淀粉酶、脂肪酶含量。
2.胰腺組織水含量檢測(cè):取1/3新鮮胰尾組織,分析天平稱濕重后置95℃烘烤48 h,稱干重,胰腺水含量為干重/濕重的百分比。
3.胰腺組織病理學(xué)檢查:取甲醛固定的胰腺組織,常規(guī)脫水、石蠟包埋、切片,HE染色,由病理科醫(yī)師盲法讀片,并參考Nevalainen和Aho[6]的標(biāo)準(zhǔn),從水腫、炎性浸潤(rùn)、出血和壞死4方面進(jìn)行病理評(píng)分。
4.胰腺組織caspase 3、caspase 8蛋白表達(dá)檢測(cè):取各組收集的胰腺組織約12.5 mg,加入含有3%蛋白酶抑制劑和1% EDTA的蛋白裂解液(Thermore公司)制備組織勻漿,4℃離心后取上清。采用BCA試劑盒(Thermore公司)定量蛋白后常規(guī)行蛋白質(zhì)印跡法檢測(cè)caspase 3、caspase 8蛋白的表達(dá),以β-actin作為內(nèi)參??筩aspase 3、caspase 8和β-actin一抗購(gòu)自CST公司,工作濃度1∶1 000。最后采用ECL發(fā)光,X片曝光、顯影、定影。應(yīng)用Bio-Rad公司的全自動(dòng)凝膠成像系統(tǒng)掃描蛋白條帶,以目的條帶與內(nèi)參條帶的灰度值比表示蛋白相對(duì)表達(dá)量。
5.胰腺細(xì)胞凋亡檢測(cè):采用脫氧核糖核酸末端轉(zhuǎn)移酶介導(dǎo)的dUTP缺口末端標(biāo)記法(TUNEL)試劑盒(Roche公司)檢測(cè)胰腺組織的細(xì)胞凋亡,嚴(yán)格按照說明書操作,以DNAase 處理的切片作陽性對(duì)照。最后在共聚焦顯微鏡下觀察,細(xì)胞核中出現(xiàn)紅色熒光為凋亡細(xì)胞。為確定紅色熒光是否定位于細(xì)胞核部位,采用DAPI染色。
三、統(tǒng)計(jì)學(xué)處理
一、小鼠血清淀粉酶、脂肪酶活性及胰腺水含量的變化
AP組小鼠血清淀粉酶、脂肪酶含量及胰腺水含量均較NC組顯著升高,AAP組又較AP組顯著升高,差異均有統(tǒng)計(jì)學(xué)意義(P<0.05或<0.01);與NC組比較,AC組小鼠血清淀粉酶顯著下降,脂肪酶活性顯著升高,差異均有統(tǒng)計(jì)學(xué)意義(P值均<0.05),而胰腺水含量差異無統(tǒng)計(jì)學(xué)意義(表1)。
組別只數(shù)血淀粉酶(U/L)血脂肪酶(U/L)胰腺水含量(%)NC組53530±259502±4270.2±3.1AC組53196±187b745±42b69.6±2.0AP組535955±4607a7346±665a78.2±1.5aAAP組553607±3848c12764±2544c85.0±3.0c
注:與NC組比較,aP<0.01;bP<0.05;與AP組比較,cP<0.01
二、胰腺病理組織學(xué)改變
NC組及AC組小鼠胰腺組織無明顯病理改變;AP組小鼠胰腺明顯水腫、少量炎細(xì)胞浸潤(rùn);AAP組胰腺壞死明顯,且伴有較多炎細(xì)胞浸潤(rùn)(圖1)。NC組及AC組胰腺病理評(píng)分為0分,AP組各個(gè)病理指標(biāo)的評(píng)分均較NC組及AC組升高,AAP組評(píng)分又較AP組顯著升高,差異均有統(tǒng)計(jì)學(xué)意義(P值均<0.05,表2)。
三、胰腺組織caspase 3、caspase 8蛋白表達(dá)量的變化
NC組 、AC組、AP組、AAP組小鼠胰腺組織caspase 3蛋白表達(dá)量分別為1.017±0.078、1.287±0.097、2.018±0.078、0.244±0.024;caspase 8蛋白表達(dá)量分別為0.829±0.010、0.599±0.074、1.270±0.080、0.145±0.015 (圖2),AP組的表達(dá)較NC組顯著增加(t=9.056,P=0.012;t=5.474 ,P=0.032),而AAP組的表達(dá)較AP組顯著下降(t=21.75,P=0.012;t=13.820 ,P=0.005),且顯著低于NC組(t=9.408,P=0.011;t=38.400,P=0.001),差異均有統(tǒng)計(jì)學(xué)意義。
圖1 NC組(1A)、AC組(1B)、AP組(1C)、AAP組(1D)小鼠胰腺組織的病理變化(HE ×100)
組別只數(shù)水腫炎細(xì)胞浸潤(rùn)出血壞死NC組50000AC組50000AP組51.6±0.21.4±0.21.2±0.21.2±0.2AAP組52.4±0.22.6±0.22.2±0.22.6±0.2t值a2.3093.4643.4644.427P值a0.04970.00850.00850.0022
注:a:APP組與AP組比較
圖2 NC組(1)、AC組(2)、AP組(3)、AAP組(4)小鼠胰腺組織caspase 3、caspase 8蛋白表達(dá)量
四、胰腺組織細(xì)胞凋亡的變化
NC組、AC組、AP組、AAP組小鼠胰腺組織的凋亡細(xì)胞數(shù)分別為1、6、214、97個(gè)/10倍視野(圖3)。AP組較NC組顯著增加(t=12.80),AAP組較AP組顯著減少(t=6.051),但仍顯著高于NC組(t=9.654),差異均有統(tǒng)計(jì)學(xué)意義(P值均<0.01)。
圖3 陽性對(duì)照組、NC組、AC組、AP組、AAP組小鼠胰腺組織內(nèi)的凋亡細(xì)胞(共聚焦顯微鏡 ×20,Merge為DAPI染色與TUNEL染色融合圖)
酒精是急、慢性胰腺炎發(fā)生的常見病因和誘因,然而酒精在胰腺炎嚴(yán)重程度中的作用尚不完全清楚。最新一項(xiàng)比較結(jié)石和酒精兩種病因在AP中作用的研究結(jié)果顯示,酒精性胰腺炎較膽源性胰腺炎具有更嚴(yán)重的臨床進(jìn)程和較差的預(yù)后,并易出現(xiàn)嚴(yán)重的并發(fā)癥,例如假性囊腫的形成等[7]。Papachristou等[8]的研究也表明酒精濫用是AP時(shí)胰腺組織發(fā)生壞死的風(fēng)險(xiǎn)因素。酒精可能是胰腺炎發(fā)生重癥化的一種原因,而重癥胰腺炎又與臨床的高死亡率明顯相關(guān)。以往的許多動(dòng)物實(shí)驗(yàn)研究結(jié)果均表明,單純的酒精喂養(yǎng)并不會(huì)引起明顯的胰腺實(shí)質(zhì)水腫、腺泡細(xì)胞壞死以及胰腺組織內(nèi)炎細(xì)胞的浸潤(rùn)等AP的病理表現(xiàn),但是酒精能夠增敏膽囊收縮素(CCK)的作用,使得生理劑量的雨蛙素(CCK類似物)也可以引起飲酒精的小鼠發(fā)生AP[9]。本研究采用飲用酒精一段時(shí)間后再誘導(dǎo)AP,結(jié)果表明單純飲用酒精并沒有引起胰腺可見的病理損傷,但能夠加重雨蛙素誘導(dǎo)的胰腺炎的嚴(yán)重程度,特別是增加了腺泡細(xì)胞的壞死和炎細(xì)胞的浸潤(rùn)。
細(xì)胞常見的死亡方式包括凋亡和壞死。壞死細(xì)胞的特征表現(xiàn)為細(xì)胞器發(fā)生腫脹、細(xì)胞膜破壞,最后引起細(xì)胞的裂解,隨后裂解的細(xì)胞通常會(huì)釋放各種活性物質(zhì)和炎癥因子從而誘發(fā)明顯的炎癥反應(yīng)瀑布[10]。相反凋亡是一種細(xì)胞的程序性死亡方式,是細(xì)胞在特定因子的作用下活化細(xì)胞內(nèi)凋亡信號(hào)通路,使細(xì)胞發(fā)生皺縮,形成凋亡小體,隨后被吞噬細(xì)胞吞噬,因而不會(huì)有細(xì)胞內(nèi)成分的泄露,不會(huì)誘發(fā)組織內(nèi)的炎癥反應(yīng)[10]。研究表明,在多種不同的實(shí)驗(yàn)?zāi)P椭?,重癥胰腺炎表現(xiàn)出明顯的腺泡細(xì)胞壞死和輕度的凋亡,而輕型胰腺炎則以腺泡細(xì)胞的凋亡為主,壞死較輕[11]。因此有研究者認(rèn)為AP時(shí)的細(xì)胞凋亡可能是一種保護(hù)機(jī)制,減輕疾病的嚴(yán)重程度。隨后一系列的實(shí)驗(yàn)結(jié)果也驗(yàn)證了通過不同機(jī)制和化學(xué)藥物促進(jìn)細(xì)胞凋亡能夠減輕胰腺的壞死程度[12-13]。天冬氨酸特異性半胱氨酸蛋白酶(caspases)家族是經(jīng)典的細(xì)胞凋亡標(biāo)志物之一。caspases激活后可以酶切底物天冬氨酸羧基端從而促進(jìn)細(xì)胞凋亡發(fā)生。已有14種caspases家族成員被認(rèn)為是凋亡的關(guān)鍵效應(yīng)酶和分子執(zhí)行者,其中與凋亡密切相關(guān)的成員包括凋亡起始的caspases 2、8、9 、10和凋亡效應(yīng)的caspases3、6 、7[14]。本研究結(jié)果顯示,AP組小鼠胰腺內(nèi)的細(xì)胞凋亡通路是活化的,而在AAP組,凋亡相關(guān)蛋白的表達(dá)和凋亡細(xì)胞均顯著降低,即細(xì)胞凋亡通路被抑制,表明AP的嚴(yán)重程度與胰腺細(xì)胞的凋亡呈負(fù)相關(guān)。
總之,本研究表明酒精的長(zhǎng)期使用可以抑制急性應(yīng)激反應(yīng)時(shí)細(xì)胞保護(hù)性凋亡通路的活化,使得以凋亡為主的腺泡細(xì)胞轉(zhuǎn)為不可逆轉(zhuǎn)性壞死。
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(本文編輯:冀凱宏)
Cell apoptosis pathway in the development of alcoholic pancreatitis in mice
GuoMin,YuPengfei,BaiBin,QiuZhaoyan,WangQian,ZhaoQingchuan,LiShujun.
StateKeyLaboratoryofCancerBiology,XijingHospitalofDigestiveDiseases,FourthMilitaryMedicalUniversity,Xian710032,China
LiShujun,Email:lishujun@fmmu.edu.cn
Objective To explore the role of cell apoptosis pathway in alcoholic pancreatitis. Methods C57BL/J mice were divided into control group(NC) and Alcohol group (AC), Acute pancreatitis group (AP) and Alcoholic acute pancreatitis group(AAP). Alcohol treatment was 10% w/v ethanol feeding for 2 d, 15% w/v ethanol for 5 d, and then 20% w/v ethanol until 13 weeks. AP model was established by the intraperitoneal injection of 50μg caerulein /kg body weight once an hour for a total of 7 times. Blood samples were collected for detecting serum amylase and lipase activity. Part pancreatic tissue was collected and the wet and dry weight were both measured to calculate the water content. The routine pathological examination of the pancreatic tissues were conducted. The expression of apoptosis associated protein caspase3 and caspase8 was determined by Western blot. And cell apoptosis was determined using TUNNEL method. Results The level of serum amylase in NC group, AC group, AP group and AAP group were(3 630±259),(3 196±187),(35 955±4607) and(53 607±3 848)U/L;the level of serum lipase were(502±41),(745±42),(7 346±665)and(12 764±2 544)U/L;the water content were(70.2±3.1)%,(69.6±2.0)%,(78.2±1.5)% and(85.0±3.0)% and (12.75±0.25);the expression of caspase3 were (1.017±0.0784),(1.287±0.097), (178±0.07785) and (0.2443±0.0243); the expression of caspase8 were (0.8289±0.0096), (0.5985±0.0735), (1.27±0.08) and (0.145±0.015);the number of apoptotic cells were 1, 6, 214,97/10 high power field. The pathological score of pancreas injure in NC group, AC group, AP group and AAP group were 0, 0,(7±0.4)and (12.8±0.3), respectively. Serum amylase, lipase, water content and pathological scores in AP group were obviously higher than those in NC group (P<0.05), which in AAP group were also obviously higher than those in AP group, and all the differences were statistically significant (allP<0.05).Compared with NC group, the expressions of apoptosis associated protein caspase3 and caspase8 and the number of apoptotic cells were obviously increased in AP group, which were obviously higher than those in AAP group, but the expression of caspase3 and caspase8 in AAP group were decreased compared with NC group, and all the differences were statistically significant(allP<0.05). Conclusions Chronic alcohol exposure may aggravate the severity of pancreatitis, and the inhibition of apoptosis pathway and the enhancement of acinar cell necrosis may be involved in this process.【Key words】 Pancreatitis, alcoholic; Ethanol; Apoptosis; MiceFund program:Foundation: National Nature Science Foundation of China(81370564); Scientific and Technological Project on Social Development of Shaanxi Province(2014SF2-11)
10.3760/cma.j.issn.1674-1935.2017.01.009
710032 西安,第四軍醫(yī)大學(xué)西京消化病醫(yī)院腫瘤生物學(xué)國(guó)家重點(diǎn)實(shí)驗(yàn)室(郭敏、余鵬飛、白檳、邱兆巖、王謙),消化ICU(趙青川、李樹鈞)
李樹鈞,Email: lishujun@fmmu.edu.cn
國(guó)家自然科學(xué)基金(81370564);陜西省社會(huì)發(fā)展攻關(guān)計(jì)劃項(xiàng)目(2014SF2-11)
2016-09-01)